Assessment of Red Cell Autoantibodies in Autoimmune Hemolytic Anemia of Warm Type by a Radioactive Anti-IgG Test1

To evaluate the applicability of a radioactive ¹²⁵I-anti-IgG test (RIAT) for the detection of small amounts of IgG antibodies on red blood cells (RBC) of patients with autoimmune hemolytic anemia of warm type (AIHA), RBC of 125 patients were studied (AIHA, n=53; Coombs'-negative AIHA, n=6; chronic cold agglutinin disease, n=7; non-immune anemias, n=59). It was found that the RBC of all cases (33/33) with a positive direct IgG antiglobulin test (DAT-IgG), but also 13 out of 20 patients with a negative DAT-IgG, but detectable complement (C3/C4), and 4 out of 6 cases with Coombs'-negative AIHA gave positive results in the direct RIAT. RBC-associated IgG was higher in the DAT-IgG positive group (n=33; x?=8.1%) than in the DAT-IgG negative group (n=26; x?3.4%). There was no correlation between hypergammaglobulinemia and RBC-associated IgG. The sensitivity of the indirect RIAT was not remarkably better as compared to the indirect antiglobulin test. The RIAT is valuable in the serology of borderline cases of AIHA.     

Red blood cell membrane-bound IgG: demonstration of antibodies in patients with autoimmune haemolytic anaemia and immune complexes in patients with rheumatic diseases

Summary. Immunoglobulin G (IgG) bound in vivo to the surfaces of red blood cells (RBC-IgG) was quantificated by an enzyme-linked immunosorbent assay (ELISA) using the cells themselves as solid phase. The method was applied on RBC from normal subjects, patients with autoimmune haemolytic anaemia (AIHA) and rheumatoid patients with and without circulating immune complexes (CIC). Small amounts of RBC-IgG were detected in normal subjects and rheumatoid patients without CIC. Fifteen out of 16 patients with AIHA had increased RBC-IgG indicating RBC sensitization with IgG antibodies, although only eight patients had a positive direct antiglobulin test (DAT) with anti-IgG. Ten out of 13 rheumatoid patients with a negative DAT and with CIC had increased RBC-IgG suggesting RBC C3 receptor-bound IC. The results provide background for further studies of the significance of RBC-IgG in health and disease.     

Carbimazole-induced immune haemolytic anaemia: role of drug-red blood cell complexes for immunization

We report on a patient who developed acute intravascular immune haemolysis while receiving carbimazole. Serological studies revealed a strongly (3+) and a discretely positive (1+) direct antiglobulin test due to C3d and IgG respectively, and a very weak IgG autoantibody in the eluates. Serum from the patient contained specific carbimazole-dependent red blood cell (RBC) antibodies which reacted with all normal human RBC in the presence of free carbimazole as well as with RBC coated with the drug either in vitro or in vivo, although carbimazole itself is not detectable in plasma after oral administration. The results provide direct evidence for the sequence of the drug-RBC-antibody interaction and show that the RBC (and not plasma proteins) function as 'carrier-like' macromolecules in the immune response.     

An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques

Summary When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group 0-test red cell panels at 37°C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.     

Mechanism of autoimmune hemolytic anemia in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a malignant clonal expansion of CD5+B lymphocytes. The CD5+B lymphocytes have been postulated to produce autoantibodies. CLL patients may demonstrate features of autoimmunity including autoimmune hemolytic anemia. However, the origin of the autoantibodies causing the hemolysis is not clear. The present studies were performed to determine whether these autoantibodies are the products of the neoplastic B-CLL clones. Immunoglobulins (Ig) were eluted from washed red blood cells (RBC) obtained from two CLL patients at the time they had autoimmune (DAT-direct antiglobulin test - positive) hemolytic anemia. The light chain phenotypes of these eluted autoantibodies were determined and found to be monotypic with exact correlation to the light chain expressed on the surface of the B-CLL clones. Elutions from RBC of DAT negative patients or normal volunteers failed to demonstrate measurable amounts of Ig. In contrast, Ig eluted from RBC obtained from SLE patients with DAT positive hemolytic anemia found to be polyclonal autoantibodies exhibiting both light chain types. Furthermore, CD5+B lymphocytes obtained from the same two CLL patients (DAT+) produce, in vitro understimulation with phorbal myristate acetate (PMA), monoclonal antibodies which react and bind to RBC. Thus these studies provide direct evidence demonstrating that the antibodies causing the autoimmune hemolytic anemia in our two CLL patients are the products of the B-CLL neoplastic clones.     

Antibody-dependent cell-mediated cytotoxicity and phagocytosis of autologous red blood cells in alphamethyldopa-induced haemolysis

7 alphamethyldopa (AMD)-treated patients with positive direct antiglobulin test (DAT) were investigated. Peripheral blood mononuclear phagocytes of 2 patients suffering from haemolysis caused lysis and phagocytosis of autologous DAT-positive red blood cells (RBC). Eluates from RBC of both patients contained antibodies of IgG1 subclass and supported antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADPh) of the patients' RBC after remission. Both cytotoxic activities were proportional to the serum concentration and to the number of attacking cells. The 5 patients without overt haemolysis did not show in vitro lysis or phagocytosis of autologous RBC. These results suggest that ADCC as well as ADPh participate in the destruction of RBC in AMD-induced haemolysis in vivo.     

Rare and transient anti‐D antibody response in D− liver transplant recipients transfused with D+ red blood cells

A retrospective analysis was conducted on 20 D^? liver transplant (LT) recipients transfused with D⁺ RBCs perioperatively and screened for RBC antibodies between 2 and 6 months later. None developed anti‐D detectable by the indirect antiglobulin test. Two patients produced weak anti‐D that reacted only with papain‐treated RBCs at 10 and 11 days without any sign of immune haemolysis. Antibodies became quickly undetectable. These data suggest an unusual pattern of alloimmunization in LT recipients with rapid, weak and transient antibody response and support the safety of transfusing D⁺ RBCs in most of D^? patients during LT surgery.     

Autoimmune haemolytic anaemia in childhood associated with non-complement binding IgM autoantibodies

Red cell bound IgM autoantibodies free of associated IgG autoantibodies and unable to activate complement have not yet been considered as the cause of autoimmune haemolytic anaemia (AIHA). We report on warm type AIHA resulting from the action of IgM autoantibodies on circulating RBC. Twelve children (eight of whom were infants) with relatively severe haemolytic anaemia were studied. Whereas the clinical findings and courses of all children appeared to be compatible with warm type AIHA, the serological findings during the haemolytic phase were atypical in that the direct antiglobulin test (DAT) was negative in 11, and positive in one case due to C3d only. The use of radiolabelled antihuman globulin antibodies showed, however, that the RBC of all the patients were sensitized with warm IgM antibodies. Elevated values of IgA and/or IgG immunoglobulins on the patients' RBC were found to be present in only two cases. Complement activation by IgM autoantibodies could not be detected in all other cases neither in vivo nor in vitro. Thus, we conclude that non-complement binding IgM autoantibodies were responsible for the AIHA in the majority of these children possibly representing an as yet unrecognized variant of warm type AIHA.     

Grand rounds: autoimmune hemolytic anemia.

Autoimmune hemolytic anemia (AHA) may be either primary (ie, "idiopathic," one third of all patients) or secondary (ie, associated with underlying illness, two thirds of all patients). A positive Coombs antiglobulin test is the most important criterion for diagnosis of AHA, and characterization of RBC coating (as to whether it is by IgG alone, by IgG plus complement, or by complement alone) may be valuable in ruling out certain underlying illnesses as causative in selected patients. Many limitations to the antiglobulin test must be kept in mind. As routinely performed, a positive result requires greater than 500 molecules of IgG per RBC. Red blood cells from normal subjects have less than 35 molecules of IgG per RBC. Up to 2% to 5% of patients with AHA will have RBC coating in the 50 to 500 molecules per cell range and may therefore have "false"-negative antiglobulin tests. Apparent failures of strength of antiglobulin reactions to correlate with severity of in vivo RBC destruction may result from technical factors or may reflect intrinsic differences among autoantibodies (eg, IgG subclass, affinity). A likely mechanism of in vivo RBC destruction in AHA has been demonstrated in immune-mediated in vitro RBC "rosette" formation about macrophages and lymphocytes bearing specific receptors for subclasses of IgG and for subcomponents of complement. Increased avidity of macrophages for coated RBCs in response to such stimulus as infection may play an additional role. Treatment involves control of underlying disease and the judicious use of adrenal steroids, splenectomy, and immunosuppressive agents. Transfusions, while life-saving in life-threatening anemia, carry substantially increased risks in AHA patients. Their use should be strictly limited.     

Donath-Landsteiner Autoimmune Hemolytic Anemia in Children

During the last 4 years, we have studied a total of 531 adults and 68 children with clinically and serologically well-defined forms of immune hemolytic anemias. Among these, Donath-Landsteiner (DL) hemolysis was the underlying disease in 22 of the 68 children (32.4%), but was not observed in adults. All children with DL hemolysis suffered from acute infections presumably of viral origin. In none of the cases was the DL hemolysis suspected clinically. Boys were more often affected than girls. The hemolytic episodes were severe, but resolved within few weeks. Serologically, all patients had a strongly positive direct antiglobulin test (DAT) using anti-C3d reagents, but a weak (n = 6) or negative (n = 16) IgG-DAT. DL hemolysins were always weak and transient, detectable with enzyme-treated red blood cells (RBC) in all, with untreated RBC in 12 of 22 sera. To explore the reason why these weak antibodies can cause extensive hemolysis in vivo, we compared the action of DL antibodies and of cold agglutinins (anti-I) on RBC by several reincubations at 4 and at 37 degrees C. The data obtained from this experiment demonstrate a stronger aggravation of hemolysis by DL than by anti-I antibodies, presumably due to low-affinity interaction between the cells and DL antibodies.     

Platelet Autoimmunity

Autoimmunity to platelets was looked for by complement fixation, antiglobulin consumption, transformation and migration-inhibition techniques. Only the migration-inhibition method gave consistently positive results. The test was positive in nine out of 14 patients with idiopathic thrombocytopenic purpura and all five patients with glandular fever.     

A prospective study to determine the safety of omitting the antiglobulin crossmatch from pretransfusion testing

Transfusion medicine laboratories routinely perform a series of pretransfusion serological tests including: ABO grouping, Rh typing, and investigation of the recipient's serum to detect antibodies against blood group antigens (antibody screen). As a final check, most laboratories also perform a crossmatch in which the recipient's serum is incubated with the donor's red cells followed by the addition of an antiglobulin reagent (antiglobulin crossmatch). The need for the antiglobulin crossmatch when the antibody screen is negative has been questioned because there are few antibodies that are detected by this test. Such antibodies are usually directed against low incidence antigens that are not expressed on the screening cells and in many cases the clinical importance of these antibodies is uncertain. For these reasons, we performed a prospective study in which patients requiring red cell transfusion had a group and screen performed. If the antibody screen was negative the antiglobulin crossmatch was omitted. Following the transfusion of the blood, the antiglobulin crossmatch was performed to look for any potential incompatibility. All patients were monitored both serologically and clinically. Over the 2-year interval of the study 9128 patients were entered. There were 8936 patients (97.9%) with a negative antibody screen and 26.9% (2404 patients) were transfused a total of 10,899 red cell concentrates. The antiglobulin crossmatch performed after the transfusion indicated that 168 red cell concentrates (1.5%) would have been incompatible if the antiglobulin crossmatch had been performed pretransfusion. These 168 red cell concentrates were transfused to 119 patients during 130 transfusion episodes (defined as all transfusions administered within 24 h). Of the 130 transfusion episodes, 79.2% (103/130) were false positive laboratory results. There were 27 transfusion episodes where the antiglobulin crossmatch on blood transfused was positive due to an IgG antibody. Even though these transfused red cell concentrates were designated incompatible by the antiglobulin crossmatch, none of the patients receiving this blood had clinical or serological evidence of haemolysis. We concluded that the antiglobulin phase of the crossmatch can be omitted from pretransfusion testing without putting patients at risk.     

Positive Antiglobulin Reactions with Thawed Deglycerolized Red Blood Cells

Abstract. Four units of thawed deglycerolized RBC were found to exhibit positive direct antiglobulin reactions and could therefore not be cross-matched. Subsequent screening of 109 randomly processed thawed RBC suspensions from the Missouri-Illinois Red Cross Blood Center discovered 5.5% with positive antiglobulin reactions, the RBC coats consisting primarily of C4b, with traces of C3d. Special studies were carried out on blood freshly obtained from donors of 3 of the original units plus 1 unit discovered on screening to have strong antiglobulin positivity. Abnormalities were demonstrable in each of the 4 donor samples; one showed a strongly positive direct antiglobulin test, sera from the other 3 donors contained elevated cold agglutinins. In vitro studies with normal RBC suspended in the 3 cold agglutinin sera (fortified with CPD) demonstrated that during the glycerolization-freezing-deglycerolization procedures, the RBC became coated primarily with C4b. It is suggested that donor sera should be screened for elevated cold agglutinins before units are assigned for frozen storage.     

An Enzyme-Linked Antiglobulin Test to Detect Red Cell Globulins after Glutaraldehyde Fixation

The purpose of this study was to develop an enzyme-linked antiglobulin test (ELAT) for IgG on RBC without the hemolysis caused by the high pH of the alkaline phosphatase reaction. This was achieved by fixing the RBC with 0.05% glutaraldehyde after attachment of the antibodies. Assays using anti-D reference standards demonstrated the sensitivity to be 1-2 ng of antibody as compared to 7.5 ng for the manual indirect antiglobulin test. The coefficients of variation of these assays ranged from 10.4 to 20.1%. The mean background absorbance at 405 nm of 105 normal RBC samples was 0.08 +/- 0.03 SD. There was an increase in sensitivity of the test after the fixed RBC were stored. Dilute glutaraldehyde stabilizes the RBC membrane and the antigen-antibody linkage resulting in a more sensitive ELAT.     

The use of platelet serotonin release as a sensitive method for detecting anti-platelet antibodies and a plasma anti-platelet factor in patients with idiopathic thrombocytopenic purpura

Summary . The method described in this paper can be used for detecting anti-platelet iso-, hetero-, and drug-antibodies and for detecting a factor in the plasma of patients with idiopathic thrombocytopenic purpura that attaches to platelets (ITP factor). Plasma from the test subject, anticoagulated with citrate, is added to a suspension of normal platelets labelled with [¹⁴C] serotonin and the amount of radioactivity (serotonin) released is measured after incubating the mixture for 45 min at 37°C. Anti-platelet antibodies and drug antibodies that fix complement as well as isoantibodies not detectable in vitro by complement fixation or agglutination can be measured by this test. Plasma from 24 of 40 patients with ITP released significantly more serotonin than control plasmas (P < 0.025). False positive results were obtained in less than 2.5% of plasmas from normal individuals, from patients with a variety of diseases and from patients with secondary thrombocytopenia. However, plasmas from two of 13 patients with systemic lupus erythematosus and normal platelet levels gave positive results in the test. The 7S gamma globulin fractions of plasmas containing isoantibodies or ITP factor as well as acid eluates from platelets which had been incubated in these plasmas gave positive serotonin-release tests.     

Detection of igg sensitization of red cells with 125I staphylococcal protein A

Most cases of immune hemolytic anemia are associated with a positive direct antiglobulin test. However, in some cases, the antiglobulin test is not sensitive enough to detect low levels of red-cell bound antibodies. This report describes a method using radiolabelled purified staphylococcal protein A which is capable of detecting IgG sensitization of red cells beyond the threshold of serologic techniques. It is less cumbersome than previously described methods and does not require antibody purification procedures. Its effectiveness was demonstrated for the detection of red-cell alloantibodies and in evaluation of patients with acquired hemolytic anemias associated with a negative direct antiglobulin test.     

Application of a High Sensitivity Aggregate-Haemagglutination Test for the Diagnosis of Autoimmune Haemolytic Anaemia with a Negative Direct Antiglobulin Test

Abstract. An aggregate-haemagglutination test has been used for determining antierythrocyte autoantibodies. The first antiglobulin variant of the test allows us to establish the presence of antibodies in 33 cases with a negative direct Coombs' test. The test II (antiantiglobulin variant) proved to be positive in 88 cases of autoimmune haemolytic anaemia (AIHA) that showed a negative direct Coombs' test and a negative antiglobulin variant. Immunoglobulin G has been revealed in the majority of AIHA patients. IgM has been determined in 1 case of a symptomatic form associated with chronic lymphocytic leukaemia. IgA has been recorded in 3 cases with an idiopathic AIHA form and in 6 cases of chronic lymphocytic leukaemia. Both types of light chains were found on the surface of erythrocytes in all cases of AIHA.     

Study of Leukopenias and Thrombocytopenias by the Direct Antiglobulin Consumption Test on Leukocytes and/or Platelets

A new test, the direct antiglobulin consumption test (direct ACT), has beendevised to be performed on the leukocytes and platelets of patients. It wasperformed in parallel with the indirect antiglobulin consumption test (indirectACT), and other serologic tests on 492 individuals comprising 24 cases of diffuse lupus erythematosus, 93 primary thrombocytopenias, 18 secondarythrombocytopenias, 48 primary leukopenias or leukothrombocytopenias, 80secondary leukopenias, 101 non-leukothrombocytopenic patients and 128 normal subjects.

A good correlation was obtained between a positive or negative result andthe presence or absence of a cytopenia in the corresponding cell series. Outof 183 thrombocytopenic patients, 42.2 per cent gave a positive result withplatelets, and out of 128 leukopenic patients, 25.8 per cent gave a positive result on leukocytes, whereas of the 299 patients with normal leukocyte andplatelet counts, 98 per cent gave negative results. The test was found positivein three categories of patients:

(1) In diffuse lupus erythematosus, tests on both leukocytes and plateletswere almost uniformly positive. The indirect ACT permits a distinctionto be made between three substances in the serum of these patients.These are antiplatelet, antileukocyte-cytoplasm and antileukocyte-nuclearsubstances.

(2) The thrombocytopenic patients were subdivided into two groups, namely primary and secondary thrombocytopenia:

(a) Out of 93 cases of idiopathic thrombocytopenic purpura, about50 per cent gave a positive result with platelets. Neither thehistory nor the clinical picture suggested any differentiation between those who gave negative results, apart from the fact thata history of infection was more frequent among the negativecases. Corticosteroid therapy did not affect the result of the test,but following splenectomy, 38 per cent of the cases become negative.

(b) Of 18 patients with a secondary thrombocytopenia, three cases(16 per cent) gave a positive result.

(3) The leukopenic and leukothrombocytopenic patients were also subdividedinto two groups:

(a) Out of 48 primary or idiopathic cases, some 50 per cent gave apositive direct ACT either on leukocytes and/or on platelets. Byusing the indirect ACT it was possible to distinguish two substances in the serum, one being antiplatelet and the other an antileukocyte cytoplasmic substance.

(b) The 80 cases of secondary leukopenia or leukothrombocytopeniagave a positive result in 15 per cent of cases with leukocytes and/or with platelets.

Of the 101 non-leukothrombocytopenic patients, only five were found togive positive tests.

All the 128 normal subjects gave negative results.

The direct ACT provides direct evidence of the presence of a -globulin,probably an auto-antibody, on the leukocytes and/or the platelets of some 99per cent of cases of diffuse lupus erythematosus, of about 50 per cent ofcases of idiopathic thrombocytopenia and idiopathic leukothromobcytopenia,and in about 15 per cent of cases of secondary thrombocytopenia and leukothrombocytopenia. No marked difference was found in the history, clinicalpicture, or hematological findings between patients giving positive and negative results.

Submitted on April 3, 1961 Accepted on August 1, 1961     

A novel method using formamide for the elution of antibodies from erythrocytes

BACKGROUND AND OBJECTIVES: Accurate identification of antibodies that sensitize red blood cells (RBCs) involves dissociating them from RBCs using an in vitro elution method that does not alter their antigen-binding properties, and analysis of the eluates against a panel of RBCs. MATERIALS AND METHODS: A method was developed that allowed efficient RBC antibody elution. Human polyclonal anti-D was used to sensitize Rh-positive RBCs, and known antigen-antibody disruptive reagents were tested using these RBCs. The best reagent conditions were optimized. Eluates made were tested and compared to results obtained with a glycine-acid-based commercial elution kit to determine efficacy. Patient samples that were positive with direct antiglobulin tests (DATs), and in vitro commercial antisera-sensitized RBCs representing clinically significant antibodies, were used for evaluating the new method. RESULTS: The formamide method was efficient at removing antibodies from RBCs. The patient samples with a positive DAT had antibodies recovered with the same specificity when compared to the acid-based technique. The length of preparation time was similar for both formamide and acid-based methods. Results of testing the eluates made from reagent RBCs sensitized with commercial antisera were distinct with antigen-positive and -negative erythrocytes. CONCLUSIONS: The formamide method compares well with acid techniques and may be an alternative choice of elution method.     

抗Sa抗体在类风湿关节炎中的意义

目的测定自身免疫性结缔组织病中抗Ｓａ抗体的阳性率，着重分析抗Ｓａ抗体在类风湿关节炎（ＲＡ）中的意义。方法从人胎盘中提取Ｓａ抗原，采用免疫印迹法，测定了４０例健康人及４７８例各种自身免疫性结缔组织病患者血清中的抗Ｓａ抗体，并分析了该抗体与ＲＡ的某些临床及实验室指标的相关性。结果抗Ｓａ抗体在各组患者中的阳性率分别为：ＲＡ为３１．９％（６１／１９１），干燥综合征为３．０％（２／６７），系统性红斑狼疮为４．３％（２／４６），白塞病、肌炎／皮肌炎、其他自身免疫性结缔组织病及正常人中均为０。研究分析表明，抗Ｓａ抗体对ＲＡ的诊断敏感性为３１．９％，特异性为９８７％，阳性预报率为９３．８％，阴性预报率为７１．３％。与抗Ｓａ抗体阴性的ＲＡ患者相比，抗Ｓａ抗体阳性的患者在关节受累、晨僵、血沉、抗核抗体、Ｘ线分期和二线药物的使用方面有显著差异。结论我们在国内首次制备了Ｓａ抗原，并建立了Ｓａ抗体的检测方法，它是一种不同于类风湿因子、对ＲＡ诊断较为特异的新型自身抗体。该抗体与疾病的严重程度相关，能否有助于ＲＡ的早期诊断和分型尚需更多病例的积累和观察。